This study was aimed to evaluate the physico-chemical properties and presence of bacteria of from soil samples
contaminated with different petroleum products which are petrol (PMS), diesel (AGO), pure kerosene (DPK) and mixed
kerosene (DPK) obtained from the tank farm of NNPC/PPMC Depot in Benin City, Nigeria. The mean count for
heterotrophic bacterial ranged from 1.83x 104±0.09 cfu/g soil contaminated with mixed DPK to 5.93x104±0.02 cfu/g for
soil contaminated with PMS. Nine (9) bacterial isolates were characterized and identified; Bacillus subtilis, Micrococcus
varians, Pseudomonas aeruginosa, Klebsiellaaerogenes, Alcaligenes sp., Corynebacterium sp., Bacillus sp., Arthrobacter sp.,
and Pseudomonas sp.Bacillussp. and Pseudomonassp. were the most occurring bacteria isolates in the four soil samples.
From the physico-chemical analysis, the pH value ranged from 6.50-6.70, electrical conductivity ranged from
14.20mhos/cm-32.50mhos/cm, moisture content from 10.20%-14.50%, carbon from 1.38%-2.97%, nitrogen from
2.37%-8.19%, 0.396%-0.950%, phosphate 1.95%-5.52% and total hydrocarbon from 125mg/kg-3,050mg/kg These
results revealed that the presence of these bacteria under the stated soil condition can enhance bioremediation of
petroleum contaminated soil. This study reveals the possible biodegradability of several petroleum products by bacteria
which will aid the bioremediation of the contaminated soil.

Activities associated with petroleum exploration,
development and production operations have local
detrimental and significant impacts on the atmosphere,
soils and sediments, surface and groundwater, marine
environment, biological diversity and sustainability at
terrestrial ecosystems in the Niger Delta [1]. Discharge of
petroleum hydrocarbon and petroleum-derived waste,
streams have caused environmental pollution, adverse
human health effects, and detrimental impact on regional
economy, socio-economy problem and degradation of
host communities in the 9 oil-producing states in the
Niger Delta region [1]. Recently, anthropogenic practices
such as industrial activities, petroleum and petroleum
derivatives (such as gasoline, diesel, and kerosene spills),
and incomplete combustion of fossil fuels have caused an
accumulation of petroleum hydrocarbons in the
environment [2]. In fact, petroleum and derivatives have a
major ecological impact on contaminated marine and
terrestrial ecosystems [2]. Many important processes
influence the destination of hydrocarbons in the
environment. Among these are sorption, volatilization,
abiotic transformation (chemical or photochemical), and
biotransformation [3]. Biodegradation of oil contaminated
soils, which exploits the ability of microorganisms to
degrade and/or detoxify organic contamination, has been
established as one of the efficient, economic, versatile and
environmentally sound treatment [4].

The presence of a high enzymatic capacity allows
microbial communities to degrade complex hydrocarbons
[5]. This capacity to modify or decompose certain
pollutants, such as petroleum, summarizes the
importance of enzymes in the bioremediation process.
Their genetic diversity contributes to the metabolic
versatility of microorganisms for the transformation of
contaminants into less-toxic final products, which are
then integrated into natural biogeochemical cycles [5].
However, appropriate environmental factors such as pH,
available nitrogen and phosphorus, Organic matter,
moisture and carbon content are essential for the
performance of these organisms. The application of
nutrients to oil contaminated site to stimulate the growth
of naturally occurring hydrocarbon utilizing bacteria for
bioremediation purpose, can greatly improve the rate of
recovering of environments contaminated with petroleum
products. Therefore this study was aimed to Isolation of
Bacteria and Physicochemical Analyses of PetroleumProducts
Contaminated Soil from NNPC/PPMC Depot,
Benin City, Nigeria.

Materials and Methods

Soil samples contaminated with petroleum products
(diesel, petrol, pure and mixed kerosene) were collected
from tank draining points from tank farm at NNPC/PPMC
Benin deport. Samples were collected at different point
from the tank farm into polyethylene bags which were
duly labelled and transported to the laboratory. All
glassware used were thoroughly washed, air dried and
sterilized using an autoclave at 121oC for 15 min.

Physic-Chemical Analysis of Soil Samples

The pH was determined electrometrically by
suspending the soil in 1: 2 (soil: 0.01M CaCl2) mixture.
The suspension of the soil was allowed to stand for 30
minutes with occasional stirring and the pH measured
with a pH meter [6]. Moisture an aluminium dish was pre
weighed (W1) using a sensitive weigh balance (State
Model). Ten (10) grams of the fresh soil sample was
transferred to the dish and weight of both the dish and
soil was noted (W2). The dish containing the soil sample
was placed in a hot air oven (State Model) at 1300C and
dried to obtain a constant weight for 24 hours. The dish
was immediately transferred to a desiccator and allowed
to cool for 30 minutes. The resultant weight was taken
(W3). The moisture content was calculated and recorded
as a percentage by weight of the respective soil sample.

Total Organic carbon

The Total Organic Carbon was determined by the
Walkley-Black titrimetric method. About 0.3 g of each soil
samples were weighed into an Erlenmyer flask with 10 ml
of K2CrO7 and 20 ml concentrationH2SO4 was added. The
mixture was gently swirled until soil and reagents were
properly mixed and were allow to stand for 30 min, 100
ml of distilled water was added and the content titrated
against standardized ferrous sulphate solution to a
reddish brown end point using ferroin as the indicator.
The total organic matter was calculated from the value
obtained for the total organic carbon [7].

Total hydrocarbon content

Hydrocarbon-utilizing bacteria population was
enumerated by spread plate technique [7]by inoculating
0.1 ml of aliquot onto sterile Mineral Salt Medium (MSM)
plates with 100μl each of petroleum products viz; diesel
(AGO), petrol (PMS), pure kerosene (DPK) and mixed
kerosene (DPK). The petroleum products used was
sterilized by filtering through Millipore filter,0.45μ
diameter and stored in sterile bottles. The petroleum
products were used as the sole carbon source to isolate
hydrocarbon-utilizing bacteria. Theplates were incubated
at 25° C for 3-5 days. Any isolate which grow on nutrient
agar plates but failed to grow on mineral salt medium
plate were confirmed as non-degraders. The isolates
which grow on both the agar plates were confirmed as
hydrocarbon utilizes. Total hydrocarbon content was
calculated as described by[8] (Figure 1).

Available phosphorus

One gramme (1.0 g) of soil was shaken for 5 minutes
with 10 ml of extracting solution containing 0.03 N NH4F
and 0.1 N HCl). The solution was filtered through
Whatman filter paper and 3 ml of the filtrate was
transferred into a test tube and 3ml of ammonium
molybdate was added. Thereafter, 5 drops of mixture of
boric acid, sodium sulphite and sodium sulphates were
added. The Phosphorus content was determined
calorimetrically [9].

Available nitrogen

One gramme (1.0 g) of the soil sample was placed into
Kjedahl digestion flask. One table of a catalyst and 20 ml
concentrated tetraoxosulphate acid was added and the
mixture was hand shaken to ensure mixing. At completion
of digestion, 10 ml distilled water was added and the
solution was filtered through a Whatman filter paper.
Nitrogen was determined calorimetrically at 625 nm [9].

Total heterotrophic bacteria

THB population was enumerated by pour plate method.
1g of the sample was aseptically transferred into 100ml of
physiological saline and transferred in a series of eight 10
fold serial dilution using physiological saline. 1ml of the
aliquot from each of the dilution of 102,104,106 and 108
was inoculated by pour plate method onto Nutrient agar
(NA) in duplicates. The plates were incubated aerobically
at room temperature for 48 – 72 hrs. The resulting
colonies were counted and recorded as colony forming
units per gram (cfu/g) of soil sample [10].

Characterization and Identification of Isolates

The enumerated bacteria were isolated and stored in
NA slants at 4°C for further identification. Primary
identification was done on the basis of colony and cell
morphology and Gram staining. Colonial examination of
the isolates was carried out to determine the type of
shape, elevation and pigmentation pattern they exhibited
[11]. Secondary identification is carried out by
performing a series of Biochemical tests,[12] (Table 1).

Results and Discussion

The physicochemical parameters are show in Table 2.
The pH of pure dual purpose kerosene (DPK) is slightly
higher (6.7) than that of premium motor spirit(PMS),
automobile oil and gas (AGO), and mixed dual purpose
kerosene (DPK) which are 6.60, 6.50, and 6.60
respectively. The conductivity of pure DPK is higher
(32.50mhos/cm) compared to the PMS, AGO and Mixed
DPK. The moisture content of pure DPK was more with
14.50% while that of PMA AGO, and Mixed DPK were
10.20%, 10.80% and 10.20% respectively. Total Carbon
content was higher in AGO (4.75%) compared to PMS,
Mixed DPK and Pure DPK which have 2.19%, 1.38% and
2.97% respectively. Total organic matter, Nitrogen and
Phosphate (8.19%, 0.95% and 5.52% respectively) were
also higher in AGO. The total heterotrophic bacteria were
higher in nutrient agar plates than in MSM which was
spread with 0.1ml of petroleum products. The total
heterotrophic bacteria count is shown in Table 3. The
population density of heterotrophic bacteria in soil
sample contaminated with PMS was 5.93×104±0.20 which
was highest compared to the soil contaminated with AGO,
pure DPK and mixed DPK with population densities of
3.53×104±0.15, 2.77x104±0.24 and 1.83x104±0.09
respectively.

Key

AGO - Automotive Gas & Oils

DPK - Dual Purpose Kerosene.

PMS - Premium Motor Spirit.Another name for gasoline.

The number of colonies on mineral salt medium is
lower when compared to the mother plate without
hydrocarbons. These results showed that the bacteria
grown on enriched medium were able to utilize the
hydrocarbon. The morphological and biochemical
characterization of the bacterial isolates obtained from
the different soil collected from different spot from the
Tank farm revealed the following isolates; Bacillussubtilis,
Micrococcusvarians, Pseudomonasaeruginosa,
Klebsiellaaerogenes, Alcaligenes sp., Corynebacterium sp.,
Bacillussp., Arthrobactersp. and Pseudomonas sp. Bacillus
subtilis, Pseudomonas aeroginosa, Bacillus sp.,
Pseudomonas sp. were present in all soil samples.
Alcaligenes sp. was isolated from pure DPK,
Micrococcussp. was present in PMS and Mixed DPK,
Klebsiellaaerogenes present in Mixed DPK, Alcaligenessp.
was present in AGO, Corynebacterium sp. Present in pure
DPK, Arthrobacter sp. was present in pure DPK, while
Pseudomonassp. was present in PMS. All the isolates are
rod shaped except Micrococcus varians which is cocci in
shape. Their cells are arranged singly except for the
Bacillus sp. which occurs in chains. The biochemical test
reveals all to be catalase positive, all negative for indole,
citrate and lactose test, and all positive for glucose test
with the exception of Arthrobactersp. Interestingly these
organisms have been implicated in hydrocarbon
degradation. Many scientists studied the petroleum
degradation by various Pseudomonas species, Bacillus
species, Micrococcussp and Alcaligenes [13-16]. Jyothi et
al. [17] also isolated Bacillus species, Micrococcus luteus,
Corynebacteriumxerosis from petroleum contaminated
soil. The physico-chemical properties and microbial loads
of soil showed that there are essential nutrients in soil
especially nitrate and phosphate necessary for microbial
growth. The microorganisms isolated were:
Bacillussubtilis, Micrococcusvarians,
Pseudomonasaeruginosa, Klebsiellaaerogenes, Alcaligenes
sp., Corynebacterium sp., Bacillussp., Arthrobactersp., and
Pseudomonassp. The presence of microorganisms in the
hydrocarbon contaminated soil samples showed that soil
indigenous microflora can metabolize crude oil or
hydrocarbons in contaminated sites.

Conclusion

The presence of microorganisms in the hydrocarbon
contaminated soil samples showed that soil indigenous
microflora can metabolize crude oil or hydrocarbons in
contaminated sites and these bacteria under the stated
soil condition can enhance bioremediation of petroleum
contaminated soil. This study reveals the possible
biodegradability of several petroleum products by
bacteria which will aid the bioremediation of the
contaminated soil. Further research in genetically
modified bacteria that will be able to degrade all types of
petroleum products which will advance the
bioremediation of contaminated soil.